The goal of the goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required, and the MS and MS-MS measurements must be very fast and reliable or throughput is insufficient for practical applications. Several separation schemes have been developed or proposed that may produce up to 100,000 fractions/day for MS and MS-MS analysis with ca. 10-20 peptides per fraction on average. MS techniques are available that can approach the speed required for the MS measurement, but no current MS-MS technique comes within an order of magnitude of the required speed. [unreadable] The new TOF-TOF will produce high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced will be interpreted with corresponding speed. The high speed is achieved by using a laser rate 25 times faster than previous MALDI instruments and multiple precursor ion selection following each laser shot. This instrument is unique in that it provides high-resolution precursor selection with MALDI MS-MS. Single isotopes can be selected and fragmented up to m/z 4000 with no detectable loss in ion transmission and less than 1% contribution from adjacent masses. This instrument also allows up to 50 fold multiplexing in MS-MS. Selected masses must differ by at least 1%, and are preferably within an order of magnitude range in intensity. This allows the generation of very high quality MS-MS spectrum at unprecedented speed. All of the peptides present in a complex peptide mass fingerprint containing a hundred or more peaks can be fragmented and identified without exhausting the sample. This allows speed and sensitivity of the MS-MS measurements to keep pace with the MS results. The combination of high-resolution precursor selection with high laser rate and multiplexing will allow high-quality, interpretable MS-MS spectra to be generated on detected peptides at the 10 attomole/?L level. [unreadable] Following completion of the human genome it was widely felt that "proteomics was the next big thing." Unfortunately, the proteomics factories spawned by this euphoria have failed to deliver on their promises and have gone out of business. The major problem is that the dynamic range is inadequate. The most abundant proteins are routinely detected by a variety of techniques, but lower abundance proteins are usually not detected, particularly if stringent conditions for confident identification are imposed. The limited dynamic range of mass spectrometry can be overcome by extensive fractionation of the sample, but this then requires that very large numbers of fractions must be analyzed. The new TOF-TOF system provides a practical solution to this problem. [unreadable] [unreadable] [unreadable]